Article

Bacteriophage cocktails as an environmentally-friendly approach to prevent Vibrio parahaemolyticus and Vibrio harveyi infections in brine shrimp (Artemia franciscana) production

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Abstract

Vibrio parahaemolyticus and Vibrio harveyi are commonly associated with brine shrimp, where they proliferate at the time of hatching and colonize nauplius surfaces, becoming a potential risk to organisms feeding on them. Among the strategies to prevent their occurrence, the use of chemotherapeutic agents and disinfectants could be considered; however, these strategies have side effects on the quality of brine shrimp and the environment. As a consequence, the use of bacteriophages (phages) is gaining interest in aquaculture. In this study, we investigated the effect of phage therapy (single phages and cocktails) on hatching and survival of brine shrimp (Artemia franciscana) cysts and nauplii exposed to pathogenic strains of V. parahaemolyticus and V. harveyi. The results revealed that application of single phages enhanced significantly (p<0.05) the hatching success and survival rate, even at low multiplicity of infection (MOI). Subsequently, these promising phages were mixed (cocktails) and administered to brine shrimp cysts and nauplii exposed to both pathogenic Vibrio species. The results also revealed an enhanced hatching success (100 % in both cases) and survival rate (from 85 to 89 %) in groups treated with phage cocktails, whereas control groups had a hatching success of around 50% and survival rate from 40 to 50%. Given this, phage cocktails offer an alternative to chemotherapeutic agents routinely used in brine shrimp production.

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... As an alternative bio-control agent, bacteriophages exhibit high host specificity and strong lytic activity (Bao et al., 2015). Phage cocktails have been used to enhance hatching and survival of brine shrimp nauplii exposed to V. parahaemolyticus, which led to an enhanced hatching success (100% in both cases) and survival rate (from 85 to 89%) in groups treated with phage cocktails (Quiroz-Guzmán et al., 2018). In addition, a newly identified phage has been applied to oyster samples to control V. parahaemolyticus growth, resulting in inactivation of 99% of V. parahaemolyticus on the oyster meat surface (Zhang et al., 2018). ...
... In another report, the survival rate (89%) in brine shrimp (A. franciscana) groups treated with phage cocktails clearly improved, whereas control groups had a survival rate of 50% (Quiroz-Guzmán et al., 2018). The phages used in the cocktail were selected taking into consideration their wide host range. ...
... Recent experimental phage therapy studies have also demonstrated positive results in controlling important bacterial fish pathogens such as Flavobacterium psychrophilum and Vibrio spp. [23][24][25][26][27]. However, phage therapy requires comprehensive knowledge of the applied bacteriophages therefore a full characterization of their biological and genomic attributes that includes their infectivity, lifestyle, stability, and possible virulence and antibiotic resistance encoding genes [28]. ...
... In the in vivo trial with Artemia nauplii, we found that a single dosage of vB_VhaM_pir03 was effective in increasing survival of Artemia nauplii infected with Vibrio harveyi strain Vh5 at 24 h post infection even at MOI 0.1. This result was showed that vB_VhaM_pir03 performed slightly better than a previously reported phage therapy trial with Artemia spp. in which the survival was measured at 50% [26] However, it was observed that vB_VhaM_pir03 was unable to provide protection to the Artemia nauplii at 48 h post infection. Nonetheless, Artemia nauplii population still showed a higher percentage of survival to the untreated population which suggested a residual effect of protection. ...
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... Phage resistance is a concerning issue in phage therapy [97] since bacteria populations have various protection mechanisms against phages [98]. The combination of different phages, phage cocktails, has been suggested as a workaround, a practice that has yielded promising results [99][100][101]. ...
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Due to the emergence of multidrug-resistant bacteria, commonly known as “superbugs”, phage therapy for the control of bacterial diseases rose in popularity. In this context, the use of phages for the management of many important bacterial diseases in the aquaculture environment is auspicious. Vibrio harveyi, a well-known and serious bacterial pathogen, is responsible for many disease outbreaks in aquaculture, resulting in huge economic and production losses. We isolated and fully characterized a novel bacteriophage, Vibrio phage Virtus, infecting V. harveyi strain VH2. Vibrio phage Virtus can infect a wide spectrum of Vibrio spp., including strains of V. harveyi, V. owensii, V. campbellii, V. parahaemolyticus, and V. mediterranei. It has a latent period of 40 min with an unusually high burst size of 3200 PFU/cell. Vibrio phage Virtus has a double-stranded DNA of 82,960 base pairs with 127 predicted open reading frames (ORFs). No virulence, antibiotic resistance, or integrase-encoding genes were detected. In vivo phage therapy trials in gilthead seabream, Sparus aurata, larvae demonstrated that Vibrio phage Virtus was able to significantly improve the survival of larvae for five days at a multiplicity of infection (MOI) of 10, which suggests that it can be an excellent candidate for phage therapy.
... El receptor de este fago se ha identificado como proteína de membrana externa K (OmpK), que generalmente está presente en el grupo Vibrio (Inoue, Matsuzaki & Tanaka 1995). Además, los vibriofagos se han aplicado con éxito en ensayos de terapia de fagos con resultados prometedores en la supervivencia de peces y crustaceos (Pereira et al., 2011;Higuera et al., 2013;Silva et al., 2014;Kalatzis et al., 2016;Kalatzis et al., 2018;Rørbo et al., 2018;Quiroz-Guzmán et al., 2018;Castillo et al., 2019;Lomeli-Ortega et al., 2020;Veyrand-Quiroz et al., 2020). Donde la mayoría de los experimentos de laboratorio in vitro se ha observado un control con los fagos de cepas de V. Abedon et al., (2011) proponen que los bacteriófagos puedan ser utilizados tanto de forma profiláctica como terapeútica, ya que tienen como objetivo mantener el balance del ambiente enfocándose solo en bacterias patógenas específicas. ...
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El langostino Macrobrachium acanthurus es una especie con el potencial para ser cultivado y en el Laboratorio de Producción Acuícola de la UNAM FES Iztacala se ha trabajado en la determinación de los requerimientos nutricionales en diferentes estadios de desarrollo de esta especie. Por ello, a continuación, presentamos algunos de los avances que hemos obtenido en los requerimientos de proteína, lípidos y carbohidratos en postlarvas, de vitaminas en las larvas, así como el uso de algunos aditivos en juveniles. Se ha logrado establecer la combinación de inclusión de 35% proteína, 15-120% lípidos y un máximo de 15% de carbohidratos. La inclusión de las vitaminas A y C permiten aumentar el porcentaje de supervivencia durante la etapa larvaria, mientras que el uso de prebióticos (fructooligosacaridos y mananoligosacaridos) podrían tener un efecto positivo en la mad uración sexual de hembras.
... Previous studies have also reported that application of phages to control Vibrio species did not have a negative impact on the microbiota associated to Artemia eggs and nauplii. Moreover, a single dose reduced the levels of Vibrio species to undetectable levels (Quiroz-Guzmán et al. 2018;Lomelí-Ortega et al. 2020). ...
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... However, infection of specimens was done though inoculation to the waters, rather than injection into the specimens, as well as flowing aquaculture sites were used in the said studies. [37][38][39] One notable characteristic of using phages in therapy is that self-replication occurs within the target host. 26 Compared with other treatments, this poses an advantage over common antimicrobial treatments as it can provide a stronger bacteriolytic response and longer protective effects. ...
... El sistema CRISPR-Cas es uno de los más estudiados ya que forma parte del sistema inmune adaptativo de las bacterias y lo utilizan para degradar el ADN del fago, no obstante, algunos fagos pueden proteger su material genético con una cubierta proteica "tipo núcleo" 61 . Finalmente, para combatir la fago-resistencia se tienen los cocteles de fagos mixtos y quorum quenching 56,62,63 . Actualidad de los fagos en acuicultura. ...
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... The CRISPR-Cas system is one of the most studied since it is part of the adaptive immune system of bacteria and use it to degrade the Phage DNA, however, some phages can protect your genetic material with a protective cover "core type" theory 61 . Finally, to fight phage-resistance you have mixed phage cocktails and quorum quenching 56,62,63 . ...
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We evaluated the efficacy of bacteriophage (phage) therapy by using a murine model of gut-derived sepsis caused by Pseudomonas aeruginosa that closely resembles the clinical pathophysiology of septicemia in humans. Oral administration of a newly isolated lytic phage strain (KPP10) significantly protected mice against mortality (survival rates, 66.7% for the phage-treated group versus 0% for the saline-treated control group; P < 0.01). Mice treated with phage also had lower numbers of viable P. aeruginosa cells in their blood, liver, and spleen. The levels of inflammatory cytokines (tumor necrosis factor alpha TNF-α, interleukin-1β [IL-1β], and IL-6) in blood and liver were significantly lower in phage-treated mice than in phage-untreated mice. The number of viable P. aeruginosa cells in fecal matter in the gastrointestinal tract was significantly lower in phage-treated mice than in the saline-treated control mice. We also studied the efficacy of phage treatment for intraperitoneal infection caused by P. aeruginosa and found that phage treatment significantly improved the survival of mice, but only under limited experimental conditions. In conclusion, our findings suggest that oral administration of phage may be effective against gut-derived sepsis caused by P. aeruginosa.
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Seven bacteriophages specific to Vibrio harveyi, the causative agent of luminous vibriosis in shrimp, were isolated from coastal aquaculture systems like shrimp farms, hatcheries and tidal creeks along the east and west coast of India. All the seven phages were found to have the typical head and tail morphology with double-stranded DNA as genetic material. Morphologically, six phages were grouped under family Siphoviridae and one under Myoviridae. These phages were further characterized with respect to host range, morphology and structural proteins. Genomic fingerprinting was carried out using restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD). Major capsid proteins of all the phages detected by SDS-PAGE were distinct from one another. All the phages were found to be highly lytic for V. harveyi and had different lytic spectrum for the large number of isolates tested. Six of the seven phages isolated had a broad lytic spectrum and could be potential candidates for biocontrol of V. harveyi in aquaculture systems.
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The bacterial strain Bacillus subtilis UTM 126 produced antimicrobial activity against pathogenic Vibrio species, including V. alginolyticus, V. parahaemolyticus, and V. harveyi. The probiotic effect of B. subtilis was tested by feeding juvenile shrimp (Litopenaeus vannamei) food supplemented with B. subtilis (10(5 )CFU/g) for 28 days before an immersion challenge with V. harveyi at 10(5 )CFU/mL for 24 h. The treatment with B. subtilis UTM 126 decreased final mortality to 18.25%, compared with 51.75% in the control group. Bacillus subtilis UTM 126 has potential applications for controlling pathogenic V. harveyi in shrimp aquaculture.
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Campylobacter jejuni is a leading cause of food-borne illness. Although a natural reservoir of the pathogen is domestic poultry, the degree of genomic diversity exhibited by the species limits the application of epidemiological methods to trace specific infection sources. Bacteriophage predation is a common burden placed upon C. jejuni populations in the avian gut, and we show that amongst C. jejuni that survive bacteriophage predation in broiler chickens are bacteriophage-resistant types that display clear evidence of genomic rearrangements. These rearrangements were identified as intra-genomic inversions between Mu-like prophage DNA sequences to invert genomic segments up to 590 kb in size, the equivalent of one-third of the genome. The resulting strains exhibit three clear phenotypes: resistance to infection by virulent bacteriophage, inefficient colonisation of the broiler chicken intestine, and the production of infectious bacteriophage CampMu. These genotypes were recovered from chickens in the presence of virulent bacteriophage but not in vitro. Reintroduction of these strains into chickens in the absence of bacteriophage results in further genomic rearrangements at the same locations, leading to reversion to bacteriophage sensitivity and colonisation proficiency. These findings indicate a previously unsuspected method by which C. jejuni can generate genomic diversity associated with selective phenotypes. Genomic instability of C. jejuni in the avian gut has been adopted as a mechanism to temporarily survive bacteriophage predation and subsequent competition for resources, and would suggest that C. jejuni exists in vivo as families of related meta-genomes generated to survive local environmental pressures.
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Vaccination is an ideal method for prevention of infectious diseases, but commercially available vaccines are still limited in the aquaculture field. Bacteriophages (phages) as specific pathogen-killers are attractive agents for treating or controlling bacterial infections. Studies on phages of fish pathogens and aquaculture phage therapy have become more popular since the late 1990s, with an increasing interest in recent years. This chapter reviews research results and discusses the potential for controlling bacterial infections in aquaculture by means of phages. Ayu (Plecoglossus altivelis) is an osmerid fish with a 1-year life cycle and is one of the most popular freshwater species for sport fishing as well as for aquaculture in Japan. Two bacterial diseases, bacterial hemorrhagic ascites caused by Pseudomonas plecoglossicida and bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum have damaged the aquaculture industry and the natural resource enhancement program of this fish species since 1990. Phage therapy against bacterial diseases of fish remains an inadequately studied topic, and further intensive investigations of various aspects of the practice are required to establish its practical feasibility in aquaculture. Phage therapy is both an old and an attractive new approach to prevent and control bacterial infections and potentially applicable to any field affected by bacterial infection, including aquaculture.
Article
Bacteriophages (or phages) are viruses which infect and kill bacteria. They are ubiquitous in the environment but are inert in humans and animals. For almost 100 years they have been used therapeutically but in the West the ready availability of antibiotics has meant that they have only been used sporadically and no commercial therapeutic products are currently available. The looming antibiotic crisis means that there is now a renewed interest in phages; a number of companies are producing nontherapeutic phage products (such as food treatment sprays), some clinical trial data are available and other trials are close to commencing. Here, I review the current state of phage therapy, with reference to the historical context and discuss why the time is now right for this forgotten cure to be revisited.
Article
Vibriosis caused by luminous Vibrio harveyi commonly contributes to poor survival in shrimp hatcheries and aquaculture ponds. Lytic bacteriophages pathogenic for V. harveyi are currently being investigated as an alternative to antibiotics to prevent vibriosis. Here, 8 bacteriophages were isolated from oysters and clams using V. harveyi strains as baiting hosts. Among these bacteriophages, 1 strain (VHP6b) identified as broadly pathogenic for 27 V. harveyi strains examined was further characterized by electron microscopy and genome sequence analysis. Phage VHP6b possessed a tail and morphology consistent with it being a member of the family Siphoviridae, and its genome and proteome were most closely related to the Vibrio phages SSP02 and MAR10. An integrase gene essential for lysogeny was not evident. The ability of bacteriophage VHP6b to protect shrimp postlarvae against vibriosis caused by V. harveyi strain VH6 was demonstrated in a model system designed to simulate typical hatchery conditions. Bacteriophage treatment improved survival of postlarvae by 40 to 60% under these conditions, so therapies based on this or other bacteriophages may be useful in shrimp hatcheries.
Article
Phage therapy is an alternative to control bacterial pathogens in aquaculture. The extensive use is apparently feasible, however it is necessary to know its potential limitations arising from the particular characteristics of the aquatic environment in terms of the dynamics of bacterial infections, in particular because under some circumstances, the direct release of phages is the unique alternative to apply the treatment to the cultures. In the present study the efficacy of phage therapy was evaluated under gnotobiotic conditions during the induced vibriosis of Artemia franciscana. Axenic brine shrimp nauplii were infected with Vibrio parahaemolyticus and treated with phage therapy, the effect of the reduction in the phage dose and the effect of delay treatment were evaluated. The vibriosis was successfully prevented by phage therapy; a single dosage of vpms1 phage was effective to eliminate the adverse effects of V. parahaemolyticus in brine shrimp (P < 0.001) and their efficacy was not affected by the reduction in the dosage, even at a multiplicity of infection of 0.45. However, its beneficial effects were compromised during the infection progress; when the application of phages was delayed, phage therapy was ineffective to control the mortality induced by V. parahaemolyticus. In conclusion, under the evaluated conditions the phage therapy was effective to prevent vibriosis in brine shrimp, however, in advanced infections their ability to control the vibriosis is limited.
Article
Strain EB 101 (ATCC 17802) is designated the type strain of Vibrio parahaemolyticus (Fujino et al) Sakazaki, Iwanami and Fukumi, and the morphological, physiological, biochemical and serological characteristics of this strain are described. The characteristics of the type strain are compared with those of 200 other strains of this species.
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The abalone production in Chile has increased considerably in recent years with no sign of tapering off. Open and semi-closed circuits in the marine water zones in the north and south of Chile are the preferred areas of culture. Coastal ecosystems are subjected to a wide variety of contaminants that generate stress that affects populations via their impacts to individuals at both physiological and genetic levels. This work investigated the genomic and cellular response of post-weaning juvenile Haliotis rufescens abalone under hatchery conditions, fed with probiotic diets, and subsequently challenged with Vibrio parahaemolyticus. The expression patterns of 16 selected genes associated with different metabolic pathways were analyzed using Real-Time PCR. Gene expression was then compared to immunological response parameters in the abalone and quantification of V. parahaemolyticus during the experimental period. Both transcriptomic and immunological analyses indicated significant alteration of physiological processes in H. rufescens correlated to exposure to the pathogenic bacteria, as well as to probiotic nutrition.
Article
The use of live feeds presents a biosecurity risk for aquaculture due to the potential for inadvertent introduction of bacterial pathogens. Reared Artemia nauplii constitute the most commonly used live feed for larval aquaculture species including fish, crustaceans, and molluscs. We therefore analyzed the localization, abundance and community structure of bacteria associated with Artemia nauplii using a combination of culture-based, microscopy-based and molecular methods. Most bacteria were localized in the nauplii gut, with external surfaces having limited bacterial colonization. The bacterial community of newly hatched nauplii was dominated by populations related to uncultured members of Gammaproteobacteria and Planctomycetales. Enrichment of Artemia nauplii for 24 h with lipid concentrates or a mixture of microalgae generally increased their bacterial load relative to newly hatched nauplii, and in one case the enrichment process shifted the bacterial community towards Vibrio spp. Antimicrobial treatment using a combination of formalin, Virkon® S and a mixture of antibiotics reduced the load of culturable bacteria as expected, but strains of the genera Vibrio, Pseudomonas, Micrococcus, Brevundimonas, Sphingomonas, and Rhizobium were isolated from treated nauplii. Surprisingly, the molecular methods revealed that the antimicrobial treatment caused a relative enrichment of DNA from Vibrio spp. in the bacterial DNA pool. This demonstrated that Vibrio spp. were relatively more resistant to the treatment than other members of the bacterial community, a fact that was not revealed by the culture-based approach. While it is unknown whether the detected DNA originated in viable cells, a low-abundant but Vibrio-dominated bacterial community would represent a substantial biosecurity risk for any hatchery. The results from this study therefore support the use of disinfected Artemia nauplii in combination with probiotics to ensure that beneficial rather than potentially pathogenic bacteria dominate the bacterial community added to rearing tanks of target larvae.
Article
The potential of bacteriophage therapy to control bacterial disease in farmed fish was tested using, as an example, furunculosis of Atlantic salmon, caused by Aeromonas salmonicida subsp. salmonicida.In vivo testing with Atlantic salmon and rainbow trout (Oncorhynchus mykiss Walbaum) showed no adverse effects, with bacteriophage generally cleared within 96 h of administration by either intraperitoneal (i.p.) injection or oral in-feed.Juvenile Atlantic salmon were administered a combination of bacteriophage O, R and B (1.9 × 108 pfu fish− 1) by i.p. injection, after they had been challenged with A. salmonicida subsp. salmonicida 78027, also by i.p. injection. The fish that were injected with bacteriophage immediately after challenge died at a significantly slower rate then those that were either not treated with bacteriophage, or treated 24 h post-challenge. However, the end result (100% mortality) was not affected.In further experiments the effects of oral (1.88 × 105 pfu g− 1 fish− 1 daily for 30 days), bath (1.04 × 105 ml− 1 daily for 30 days) and i.p. (6.25 × 107 pfu fish− 1) phage treatment to control furunculosis in experimentally infected Atlantic salmon were compared with antibiotherapy (treatment with 10 mg kg− 1 bw− 1 day− 1 oxolinic acid for 10 days), using an indirect cohabitation challenge. No protection was offered by any of the bacteriophage treatments, compared to the positive challenge group, although significant protection was offered by the oxolinic acid treatment. Analysis of samples taken from the trials demonstrated that bacteriophage were correctly administered to the fish and, on occasion, were isolated from fish that had succumbed to furunculosis. It was also shown that bacteriophage resistant A. salmonicida subsp. salmonicida isolates could be recovered from mortalities in all the treatment groups.The results suggest that, although there were no safety problems associated with the approach, furunculosis in Atlantic salmon is not readily controllable by application of bacteriophage.
Article
Bacteriophages are regarded as natural antibacterial agents in food since they are able to specifically infect and lyse food-borne pathogenic bacteria without disturbing the indigenous microbiota. Two Staphylococcus aureus obligately lytic bacteriophages (vB_SauS-phi-IPLA35 and vB_SauS-phi-SauS-IPLA88), previously isolated from the dairy environment, were evaluated for their potential as biocontrol agents against this pathogenic microorganism in both fresh and hard-type cheeses. Pasteurized milk was contaminated with S. aureus Sa9 (about 10(6) CFU/mL) and a cocktail of the two lytic phages (about 10(6) PFU/mL) was also added. For control purposes, cheeses were manufactured without addition of phages. In both types of cheeses, the presence of phages resulted in a notorious decrease of S. aureus viable counts during curdling. In test fresh cheeses, a reduction of 3.83 log CFU/g of S. aureus occurred in 3h compared with control cheese, and viable counts were under the detection limits after 6h. The staphylococcal strain was undetected in both test and control cheeses at the end of the curdling process (24 h) and, of note, no re-growth occurred during cold storage. In hard cheeses, the presence of phages resulted in a continuous reduction of staphylococcal counts. In curd, viable counts of S. aureus were reduced by 4.64 log CFU/g compared with the control cheeses. At the end of ripening, 1.24 log CFU/g of the staphylococcal strain was still detected in test cheeses whereas 6.73log CFU/g was present in control cheeses. Starter strains were not affected by the presence of phages in the cheese making processes and cheeses maintained their expected physico-chemical properties.
Article
Bacteriophage ΦCJ07 with broad host ranges for Salmonella strains isolated from sewage effluent were used to reduce Salmonella Enteritidis (SE) infection in chickens. One-day-old chicks challenged with 5×10(7) colony-forming units/bird of SE were cohabitated with contact chicks and treated with three concentrations (10(5), 10(7) and 10(9) plaque forming units (PFU)/g) of bacteriophage prepared as a feed additive for 21days after challenge. Salmonella in the intestine was quantified and environmental contamination level was examined at 1, 2 and 3weeks after challenge. All treatments reduced intestinal SE colonization in challenged and contact chickens and reduced the environmental contamination level, but the reductions produced by 10(7) and 10(9)PFU/g of bacteriophage were significant (P<0.05) as compared with untreated controls. In addition, seven out of 10 (70%) contact chickens treated with 10(9)PFU/g of bacteriophage had no detectable intestinal Salmonella at 3weeks after treatment, suggesting that bacteriophage therapy significantly prevented the horizontal transmission of SE. These results provide important insights into preventive and control strategies against SE infection in poultry and indicate that the use of bacteriophage could reduce the incidence of Salmonella food poisoning.
Article
The main goal of this study was to evaluate the efficiency of phage therapy against one of the most common multidrug-resistant (MDR) agents of skin infections, Pseudomonas aeruginosa. A phage suspension [10(8) plaque-forming units (PFU) mL(-1)] was obtained using the clinical strain P. aeruginosa 709 as the host. The ability of the phage to inactivate P. aeruginosa was evaluated in vitro and ex vivo (human skin), using a multiplicity of infection (MOI) of 0.5 to 50. In the presence of the phage, the density of P. aeruginosa 709 [10(5) colony-forming units (CFU) mL(-1)] in the human skin decreased by 4 logs after 2 h of incubation. The application of a second dose of phage did not increase the efficiency of the therapy. This study indicates that the topical application of phage PA709 efficiently inactivates MDR P. aeruginosa 709. The high efficiency in the inactivation of MDR P. aeruginosa 709, its considerable host range (infection of 30 % of the P. aeruginosa isolates) and its high stability in buffer and ex vivo human skin make this phage very promising for the treatment of P. aeruginosa skin infections. The phage-bacteria interactions were examined in vitro and in ex vivo in order to provide a basis for the selection of the most suitable protocol for subsequent in vivo experiments.
Article
Since no artificial feed formulation is yet available to completely substitute for Artemia, feeding live prey to young fish larvae still remains essential in commercial hatchery operations. The nutritional quality of commercially available Artemia strains being relatively poor in eicosapentaenoic acid (EPA, 20:5n-3) and especially docosahexaenoic acid (DHA, 22:6n-3), it is essential and common practice to enrich these live prey with emulsions of marine oils.In Artemia, the most commonly applied boosting technique is a 24-h enrichment period after hatching. However, the variability of enrichment studied in one Artemia strain (Great Salt Lake, Utah, USA) by the ICES Working Group on Mass Rearing of Juvenile Fish, showed a high variability in fatty acid bioaccumulation under laboratory or commercial conditions. To avoid the variation originating from differences in commercial preparations, standardized ICES emulsions with different HUFA and DHA/EPA ratios have been formulated and are available for research purposes. It should be emphasized, however, that the enrichment technique has limitations as Artemia are selectively catabolizing some of the nutrients such as DHA and phospholipids. Research on the kinetics of DHA catabolism in various Artemia strains has shown that DHA catabolism is strain-dependent and could partially be overcome by the use of strains of different geographical origin.Nowadays, various enrichment emulsions have been formulated differing in the fatty acid composition of their triglycerides. In this respect, the traditional formulations rich in EPA have been replaced by new products rich in DHA and arachidonic acid. To reduce the risks for oxidation of these fatty acids, higher concentrations of vitamin E are incorporated into the emulsions. Also, vitamin C has been incorporated in booster formulations that increase the level of ascorbic acid in Artemia to 2000 ppm.All these changes in the formulation of the enrichment diets offer more possibilities to cover the needs of different species and help to reduce problems related to diseases, stress resistance, malformation, and pigmentation in numerous fish species.Although continuous disinfection of Artemia during hatching and enrichment is becoming a routine operation in many hatcheries, the interference of bacteria in hatching and enrichment remains an important study object for which probionts might also give some solutions.As more attention is given to the use of on-grown Artemia as a cheaper alternative to the use of nauplii, simple cost-effective production techniques have been developed. The use of the right size of on-grown Artemia for feeding ensures a better energy balance in food intake and assimilation, thereby improving the performance of the fish. Furthermore, its palatability induces a good and fast feeding response. These characteristics, coupled with the use of bioencapsulation techniques to enhance the quality of the on-grown Artemia, make this organism an optimum diet for nursery of the fish.
Article
Multidrug-resistant Salmonella Typhimurium U288 is a significant pathogen of pigs, accounting for over half of all outbreaks on UK pig production premises. The potential of this serovar, and other salmonellae, to enter the food chain during the slaughtering process requires that efforts be made to reduce the prevalence of these bacteria at both the pre- and post-harvest stages of production. A bacteriophage cocktail (PC1) capable of lysing various Salmonella enterica serovars was designed using the broad host-range phage Felix 01, and three phages isolated from sewage. PC1 applied to pig skin experimentally-contaminated with U288 achieved significant reductions (P<0.05) in Salmonella counts when stored at 4 °C over 96 h. Reductions of >1 log₁₀ unit were observed when the ratio of phage applied was in excess of the bacterial concentration. The treatment was found to be effective at a multiplicity of infection (MOI) of 10 or above, with no significant reductions taking place when the MOI was less than 10. Under these conditions U288 counts of log₁₀ 4.1-4.3 CFU were reduced to undetectable levels following the application of PC1 to pig skin (>99% reduction). These data suggest phage cocktails could be employed post-slaughter as a means to reduce Salmonella contamination of pig carcasses.
Article
This study determined the effect of a previously characterized phage mixture, referred as BEC8 on enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains applied on materials typically used in food processing surfaces. Sterile stainless steel chips (SSC), ceramic tile chips (CTC), and high density polyethylene chips (HDPEC) were used. Cultures of EHEC O157:H7 strains EK27, ATCC 43895, and 472 were combined, spot inoculated on surfaces, and dried. Chips were inoculated with 10(6), 10(5), and 10(4)CFU/chip, to obtain 1, 10 and 100 multiplicity of infection (MOI) values, respectively. Controls and BEC8 (approx. 10(6) PFU/chip) were applied on inoculated surfaces and incubated at 4, 12, 23, and 37 °C. EHEC survival was determined using standard plate count on tryptic soy agar. At 37 °C and 12 °C on SSC, no survivors were detected (detection limit 10CFU/chip) after BEC8 treatment at MOI of 100 after 10 min and at 23 °C after 1h on SSC. A similar result was obtained on CTC at 37 °C after 10 min, and after 1h at 23 °C. These results indicated that the phage cocktail was effective within an hour against low levels of the EHEC mixture at above room temperature on all 3 hard surfaces.
Article
Infections caused by avian pathogenic Escherichia coli (APEC) cause important economic losses to poultry industry. The studies presented herein, aimed at investigating the in vivo performance of a cocktail of three phages in treating severe respiratory E. coli infections in experimentally contaminated birds and naturally infected flocks. Three lytic coliphages, phi F78E (Myoviridae), phi F258E (Siphoviridae) and phi F61E (Myoviridae) were combined in a 5.0 × 10(7)PFU/ml cocktail to be used in naturally APEC infected flocks (refractive to antibiotherapy). Experimentally infected birds were treated with phi F78E at two different titres (10(7)PFU/ml and 10(9)PFU/ml). Phage administration was performed orally and by spray, in a single application. The morbidity, mortality and pathology scores were compared with control birds not receiving phage therapy. The results revealed that the success of phage therapy in experimental rooms was dosage dependent, being 10(7)PFU/ml not enough to treat the infected chickens whereas a concentration of 10(9)PFU/ml of phi F78E allowed a decrease of 25% and 43% in chickens' mortality and morbidity, respectively. In the large scale experiments, the results obtained showed a remarkable efficacy of the low titre phage cocktail (10(7)PFU/ml) in decreasing the flocks' mortality to levels below 0.5% in no more than 3 weeks, with no recidivism. Based on the results we can conclude that phage treatment is a valuable alternative to control APEC infections in poultry.
Article
Phages are now acknowledged as the most abundant microorganisms on the planet and are also possibly the most diversified. This diversity is mostly driven by their dynamic adaptation when facing selective pressure such as phage resistance mechanisms, which are widespread in bacterial hosts. When infecting bacterial cells, phages face a range of antiviral mechanisms, and they have evolved multiple tactics to avoid, circumvent or subvert these mechanisms in order to thrive in most environments. In this Review, we highlight the most important antiviral mechanisms of bacteria as well as the counter-attacks used by phages to evade these systems.
Article
Microbial biofilms developing in aquaculture tanks represent a reservoir for opportunistic bacterial pathogens, and procedures to control formation and bacterial composition of biofilms are important for the development of commercially viable aquaculture industries. This study investigated the effects of seawater ozonation on biofilm development on microscope glass slides placed in small-scale aquaculture tanks containing the live feed organism Artemia. Fluorescence in situ hybridization (FISH) demonstrated that ozonation accelerated the biofilm formation cycle, while it delayed the establishment of filamentous bacteria. Gammaproteobacteria and Alphaproteobacteria were the most abundant bacterial groups in the biofilm for both water types, but ozonation influenced their dynamics. With ozonation, the bacterial community structure was relatively stable and dominated by Gammaproteobacteria throughout the experiment (21-66% of total bacteria). Without ozonation, the community showed larger fluctuations, and Alphaproteobacteria emerged as dominant after 18 days (up to 54% of total bacteria). Ozonation of seawater also affected the dynamics of less abundant populations in the biofilm such as Betaproteobacteria, Planctomycetales and the Cytophaga/Flavobacterium branch of phylum Bacteroidetes. The abundance of Thiothrix, a bacterial genus capable of filamentous growth and fouling of larvae, increased with time for both water types, while no temporal trend could be detected for the genus Vibrio. Denaturing gradient gel electrophoresis (DGGE) demonstrated temporal changes in the dominant bacterial populations for both water types. Sequencing of DGGE bands confirmed the FISH data, and sequences were related to bacterial groups commonly found in biofilms of aquaculture systems. Several populations were closely related to organisms involved in sulfur cycling. Improved Artemia survival rates in tanks receiving ozonated water suggested a positive effect of ozonation on animal health. Although the used ozonation protocol did not hinder biofilm formation, the results suggest ozonation as a promising approach for manipulation of bacterial populations in aquaculture systems, which can prove beneficial for cultured animals.
Article
Data from the World Health Organization confirm a decrease in the effectiveness of antibiotic therapy. The spread of bacteria resistant to several groups of antibiotics creates more problems in the treatment of various diseases, especially in children. It is possible that pharmacological agents may prove to be ineffective in curing infections caused by resistant pathogens, and this could lead to a post-antibiotic era. It is necessary to extend the arsenal of the available therapeutic tools. Bacteriophages have long been used therapeutically and prophylactically in children. In the beginnings of phage therapy, enthusiasm prevailed over the rational methods used in contemporary controlled studies. Many people dealing with phages described cases of successful therapy, but did not conduct comparative studies. Nevertheless, phage administration seems to be safe, even in children after intravenous administration. The therapeutic and prophylactic application of phages is now experiencing a renaissance of interest. The authors' own recent analysis demonstrated the cost effectiveness of phages over antibiotic especially in the treatment of infections caused by multidrug-resistant bacteria. It can be concluded that the results of the therapeutic and prophylactic application of phages against multi-drug resistant pathogens are encouraging. It seems clear that bacteriophages need further evaluation regarding the control of bacterial infection in children.
Article
The specter of antibiotic-resistant bacteria has provoked renewed interest in the possible use of bacteriophages to control bacterial infections. We argue that clinical application of phage therapy has been held back by a failure to appreciate the extent to which the pharmacokinetics of self-replicating agents differ from those of normal drugs. For self-replicating pharmaceutical agents, treatment outcome depends critically on various density-dependent thresholds, often with apparently paradoxical consequences. An ability to predict these thresholds and associated critical time points is a necessity if phage therapy is to become clinically practicable.
Article
Nature Biotechnology journal featuring biotechnology articles and science research papers of commercial interest in pharmaceutical, medical, and environmental sciences.
Article
Phage therapy is complicated by the self-replicating nature of phage. It is difficult to extrapolate from in vitro phage growth data to in vivo expectations, difficult to interpret in vivo data and difficult to generalize from one in vivo situation to another. Various generic models of phage growth have been used as the theoretical basis for understanding the kinetics of phage therapy. Here, we have experimentally tested the efficacy of such simple models to predict, qualitatively and quantitatively, the growth of phage and the phage proliferation threshold in vitro. Naturally occurring, antibiotic-resistant bacteria were used to measure the growth of phage in vivo. In homogenous, in vitro environments, the models were predictive of T4 phage growth on Escherichia coli RR1. However, the models were not able to predict growth of T4 phage or K1-5 phage in the more complex environment of the rat's digestive tract. To explore fully the kinetics of phage therapy, more complex models need to be devised. We suggest that it may be necessary to consider and model the interactions between phage growth parameters and bacterial growth parameters.
Article
Lysis-deficient (LyD) bacteriophages (phages) kill bacteria without endotoxin (Et) release. This may minimize systemic cytokine responses and limit inflammation in bacterial sepsis. We determined the effects of t amber A3 T4 LyD and virulent wild-type (WT) phages on mouse bacterial peritonitis. Balb/c mice were injected with B40sul Escherichia coli, treated intraperitoneally with LyD, WT, or a beta-lactam antibiotic [latamoxef sodium (LMOX)], and followed for survival. We measured Et release, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, as well as bacterial counts and peritoneal exudative cells (PECs) in peritoneal lavage fluid at 6 and 12 hours after infection. LyD mice showed significantly greater survival compared with other groups. Et levels were significantly lower in the LyD mice at 6 and 12 hours after infection. TNF-alpha and IL-6 levels were lower in LyD mice compared with control (untreated) mice at 12 hours. Compared with controls, bacteria counts in peritoneal lavage fluid were lower in all treatment groups (LyD, WT, or LMOX) at 6 and 12 hours. PEC counts were highest in LyD mice at 6 hours but significantly lower than that in WT phage- and LMOX-treated mice at 12 hours. LyD phage therapy significantly improves survival and attenuates the systemic effects of bacterial sepsis by minimizing Et release and pro-inflammatory mediators in murine bacterial peritonitis. Further studies may find phage therapy useful in treating peritonitis and multidrug-resistant bacterial infections.
Article
The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days, starting 5 days after C. jejuni colonization of the broilers had been established. Treatment was monitored by enumerating Campylobacter colony forming units (CFU) and phage plaque forming units (PFU) from caecal content. Counts were compared with control birds not receiving phage therapy. A clear 3 log decline in C. jejuni counts was initially observed in the therapeutic group, however, after 5 days bacterial counts stabilized at a level 1 log lower than that of the control group. Colonization of C. jejuni in the prevention group was delayed by the treatment and after an initial 2 log reduction, colonization stabilized within a week at levels comparable to the therapeutic group. The CFU and PFU counts displayed opposing highs and lows over time, indicative of alternating shifts in amplification of bacteria and phages. There were no adverse health effects from the phage treatment. Two different phages were combined as therapeutic treatment of Campylobacter positive chickens challenged at the age approaching broiler harvest. This again resulted in a significant decrease in Campylobacter colonization. We conclude that phage treatment is a promising alternative for reducing C. jejuni colonization in broilers.
Article
The bacteriophage Esc-A was isolated from sewage by using the intestinal pathogenic Escherichia coli 3-1 as the host. Toxicity in chickens showed its safety as a bio-product. Phage therapy against diarrhea in chickens indicated that Esc-A could decrease the death rate more efficiently compared with antibiotic treatments.
Article
Recent examples of the use of bacteriophages in controlling bacterial infections are presented, some of which show therapeutic promise. The therapeutic use of bacteriophages, possibly in combination with antibiotics, may be a valuable approach. However, it is also quite clear that the safe and controlled use of phage therapy will require detailed information on the properties and behavior of specific phage-bacterium systems, both in vitro and especially in vivo. In vivo susceptibility of bacterial pathogens to bacteriophages is still largely poorly understood and future research on more phage-bacterium systems has to be undertaken to define the requirements for successful phage treatments.
Article
The emergence of mycobacteria resistant to currently available antimicrobial agents has become an important problem in modern medicine. Mycobacterium avium and M. tuberculosis are intracellular pathogens that replicate and survive within the mononuclear phagocytes. TM4 is a lytic mycobacteriophage that kills both extracellular M. avium and M. tuberculosis. When delivered by M. smegmatis transiently infected with TM4, it kills both M. avium and M. tuberculosis within RAW 264.7 macrophages. To evaluate the treatment of M. avium infection with phage in vivo, C57 BL/6 mice were infected with M. avium 109 and, 7 days later, treated either once or twice with TM4 phage (7.9 x 10(10) PFU/ml), M. smegmatis (4 x 10(8) cFU/ml), or M. smegmatis with TM4 phage delivered intravenously (i.v.). Treatment with TM4 phage alone or M. smegmatis without TM4 did not show a significant decrease in number of intracellular bacteria in the spleen compared with untreated control. In contrast, administration of M. smegmatis-TM4 resulted in a significant decrease in the number of M. avium in the spleen. However, 23% of bacteria recovered from treated mice were resistant to TM4. These in vivo studies confirmed the in vitro findings that an avirulent mycobacterium can be used as a carrier to deliver antimycobacterial phage intracellularly.
Article
A simple microplate method, based on conversion of tetrazolium to formazan, was devised for rapidly assessing Salmonella survival after phage treatment. Results were easily interpretable. Monitoring with a microplate reader was useful, but not required. The method was used in defining phage-Salmonella interactions for selection of phage biocontrol cocktails.
Inhibitory activity of probiotic Bacillus subtilis UTM 126
  • J L Balcázar
  • T Rojas-Luna
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